Benzazepinone alpha vintegrin receptor antagonists

ABSTRACT

The present invention relates to novel benzazepinone derivatives, their synthesis, and their use as αv integrin receptor antagonists. More particularly, the compounds of the present invention are antagonists of the integrin receptors αvβ3 and αvβ5 and are therefore useful for inhibiting bone resorption, treating and/or preventing osteoporosis, and inhibiting vascular restenosis, diabetic retinopathy, macular degeneration, angiogenesis, atherosclerosis, inflammatory arthritis, cancer, and metastatic tumor growth.

FIELD OF THE INVENTION

[0001] The present invention relates to benzazepinone derivatives, theirsynthesis, and their use as αv integrin receptor antagonists. Moreparticularly, the compounds of the present invention are antagonists ofthe integrin receptors αvβ3, αvβ5, and αv integrin receptors associatedwith other β-subunits, and are useful for inhibiting bone resorption,treating and/or preventing osteoporosis, and inhibiting vascularrestenosis, diabetic retinopathy, macular degeneration, angiogenesis,atherosclerosis, inflammatory arthritis, cancer, and metastatic tumorgrowth.

BACKGROUND OF THE INVENTION

[0002] It is believed that a wide variety of disease states andconditions can be mediated by acting on integrin receptors and thatintegrin receptor antagonists represent a useful class of drugs.Integrin receptors are heterodimeric transmembrane receptors throughwhich cells attach and communicate with extracellular matrices and othercells. (See S. B. Rodan and G. A. Rodan, “Integrin Function InOsteoclasts,” Journal of Endocrinology, 154: S47-S56 (1997), which isincorporated by reference herein in its entirety).

[0003] In one aspect of the present invention, the compounds disclosedherein are useful for inhibiting bone resorption. Bone resorption ismediated by the action of cells known as osteoclasts. Osteoclasts arelarge multinucleated cells of up to about 400 mm in diameter that resorbmineralized tissue, chiefly calcium carbonate and calcium phosphate, invertebrates. Osteoclasts are actively motile cells that migrate alongthe surface of bone, and can bind to bone, secrete necessary acids andproteases, thereby causing the actual resorption of mineralized tissuefrom the bone. More specifically, osteoclasts are believed to exist inat least two physiological states, namely, the secretory state and themigratory or motile state. In the secretory state, osteoclasts are flat,attach to the bone matrix via a tight attachment zone (sealing zone),become highly polarized, form a ruffled border, and secrete lysosomalenzymes and protons to resorb bone. The adhesion of osteoclasts to bonesurfaces is an important initial step in bone resorption. In themigratory or motile state, the osteoclasts migrate across bone matrixand do not take part in resorption until they again attach to bone.

[0004] Integrins are involved in osteoclast attachment, activation andmigration. The most abundant integrin on osteoclasts, e.g., on rat,chicken, mouse and human osteoclasts, is an integrin receptor known asαvβ3, which is thought to interact in bone with matrix proteins thatcontain the RGD sequence. Antibodies to αvβ3 block bone resorption invitro indicating that this integrin plays a key role in the resorptiveprocess. There is increasing evidence to suggest that αvβ3 ligands canbe used effectively to inhibit osteoclast mediated bone resorption invivo in mammals.

[0005] The current major bone diseases of public concern areosteoporosis, hypercalcemia of malignancy, osteopenia due to bonemetastases, periodontal disease, hyperparathyroidism, periarticularerosions in rheumatoid arthritis, Paget's disease,immobilization-induced osteopenia, and glucocorticoid-inducedosteoporosis. All of these conditions are characterized by bone loss,resulting from an imbalance between bone resorption, i.e. breakdown, andbone formation, which continues throughout life at the rate of about 14%per year on the average. However, the rate of bone turnover differs fromsite to site; for example, it is higher in the trabecular bone of thevertebrae and the alveolar bone in the jaws than in the cortices of thelong bones. The potential for bone loss is directly related to turnoverand can amount to over 5% per year in vertebrae immediately followingmenopause, a condition which leads to increased fracture risk.

[0006] In the United States, there are currently about 20 million peoplewith detectable fractures of the vertebrae due to osteoporosis. Inaddition, there are about 250,000 hip fractures per year attributed toosteoporosis. This clinical situation is associated with a 12% mortalityrate within the first two years, while 30% of the patients requirenursing home care after the fracture.

[0007] Individuals suffering from all the conditions listed above wouldbenefit from treatment with agents which inhibit bone resorption.

[0008] Additionally, αvβ3 ligands have been found to be useful intreating and/or inhibiting restenosis (i.e. recurrence of stenosis aftercorrective surgery on the heart valve), atherosclerosis, diabeticretinopathy, macular degeneration, and angiogenesis (i.e. formation ofnew blood vessels), and inhibiting viral disease. Moreover, it has beenpostulated that the growth of tumors depends on an adequate bloodsupply, which in turn is dependent on the growth of new vessels into thetumor; thus, inhibition of angiogenesis can cause tumor regression inanimal models (See Harrison's Principles of Internal Medicine, 12th ed.,1991, which is incorporated by reference herein in its entirety).Therefore, αvβ3 antagonists which inhibit angiogenesis can be useful inthe treatment of cancer by inhibiting tumor growth (See, e.g., Brooks etal., Cell, 79:1157-1164 (1994), which is incorporated by referenceherein in its entirety).

[0009] Evidence has also been presented suggesting that angiogenesis isa central factor in the initiation and persistence of arthritic disease,and that the vascular integrin αvβ3 may be a preferred target ininflammatory arthritis. Therefore, αvβ3 antagonists which inhibitangiogenesis may represent a novel therapeutic approach to the treatmentof arthritic disease, such as rheumatoid arthritis (see C. M. Storgard,et al., “Decreased angiogenesis and arthritic disease in rabbits treatedwith an αvβ3 antagonist,” J. Clin. Invest., 103: 47-54 (1999);“Cilengitide,” Drugs of the Future, 25: 674-678 (2000); and A. M.Badger, et al., “Disease-Modifying Activity of SB 273005, An OrallyActive, Nonpeptide αvβ3 (Vitronectin Receptor) Antagonist, in RatAdjuvant-Induced Arthritis,” Arthritis & Rheumatism, 44: 128-137 (2001);each of which is incorporated by reference herein in its entirety).

[0010] Moreover, the compounds of this invention can also inhibitneovascularization by acting as an antagonist of the integrin receptorαvβ5. A monoclonal antibody for αvβ5 has been shown to inhibitVEGF-induced angiogenesis in rabbit cornea and the chick chorioallantoicmembrane model (See M. C. Friedlander, et al., Science 270: 1500-1502(1995), which is incorporated by reference herein in its entirety).Thus, compounds that antagonize αvβ5 are useful for treating andpreventing macular degeneration, diabetic retinopathy, cancer, andmetastatic tumor growth.

[0011] Additionally, the compounds of the instant invention can inhibitangiogenesis and inflammation by acting as antagonists of αv integrinreceptors associated with other β subunits, suh as αvβ6 and αvβ8 (See,for example, Melpo Christofidou-Solomidou, et al., “Expression andFunction of Endothelial Cell αv Integrin Receptors in Wound-InducedHuman Angiogenesis in Human Skin/SCID Mice Chimeras,” American Journalof Pathology, 151: 975-83 (1997) and Xiao-Zhu Huang, et al.,“Inactivation of the Integrin β6 Subunit Gene Reveals a Role ofEpithelial Integrins in Regulating Inflammation in the Lungs and Skin,”Journal of Cell Biology, 133: 921-28 (1996), which are incorporated byreference herein in their entirety).

[0012] In addition, the compounds of this invention can also antagonizeboth the αvβ3 and αvβ5 receptors and is therefore useful for inhibitingbone resorption, treating and preventing osteoporosis, and inhibitingvascular restenosis, diabetic retinopathy, macular degeneration,angiogenesis, atherosclerosis, inflammatory arthritis, cancer, andmetastatic tumor growth.

[0013] Peptidyl as well as peptidomimetic antagonists of the αvβ3integrin receptor have been described both in the scientific and patentliterature. For example, reference is made to W. J. Hoekstra and B. L.Poulter, Curr. Med. Chem. 5: 195-204 (1998) and references citedtherein; WO 95/32710; WO 95/37655; WO 97/01540; WO 97/37655; WO98/08840; WO 98/18460; WO 98/18461; WO 98/25892; WO 98/31359; WO98/30542; WO 99/15506; WO 99/15507; WO 00/03973; EP 853084; EP 854140;EP 854145; U.S. Pat. Nos. 5,204,350; 5,217,994; 5,639,754; 5,741,796;5,780,426; 5,929,120; 5,952,341; 6,017,925; and 6,048,861. Evidence ofthe ability of αvβ3 integrin receptor antagonists to prevent boneresorption in vitro and in vivo has been presented (see V. W. Englemanet al., “A Peptidomimetic Antagonist of the αvβ3 Integrin Inhibits BoneResorption In Vitro and Prevents Osteoporosis In Vivo,” J. Clin. Invest.99: 2284-2292 (1997); S. B. Rodan et al., “A High Affinity Non-Peptideαvβ3 Ligand Inhibits Osteoclast Activity In Vitro and In Vivo,” J. BoneMiner. Res. 11: S289 (1996); J. F. Gourvest et al., “Prevention ofOVX-Induced Bone Loss With a Non-peptidic Ligand of the αvβ3 VitronectinReceptor,” Bone 23: S612 (1998); M. W. Lark et al., “An Orally ActiveVitronectin Receptor αvβ3 Antagonist Prevents Bone Resorption In Vitroand In Vivo in the Ovariectomized Rat,” Bone 23: S219 (1998)).

[0014] The αvβ3 integrin receptor recognizes the Arg-Gly-Asp (RGD)tripeptide sequence in its cognate matrix and cell surface glycoproteins(see J. Samanen, et al., “Vascular Indications for Integrin αvAntagonists,” Curr. Pharmaceut. Design 3: 545-584 (1997)). A benzazepinenucleus has been employed among others by Genentech and SmithKlineBeecham as a conformationally constrained Gly-Asp mimetic to elaboratenonpeptide αvβ3 integrin receptor antagonists substituted at theN-terminus with heterocyclic arginine mimetics (see R. M. Keenan et al.,“Discovery of Potent Nonpeptide Vitronectin Receptor (αvβ3)Antagonists,” J. Med. Chem. 40: 2289-2292 (1997); R. M. Keenan et al.,“Benzimidazole Derivatives As Arginine Mimetics in 1,4-BenzodiazepineNonpeptide Vitronectin Receptor (αvβ3) Antagonists,” Bioorg. Med. Chem.Lett. 8: 3165-3170 (1998); and R. M. Keenan et al., “Discovery of anImidazopyridine-Containing 1,4-Benzodiazepine Nonpeptide VitronectinReceptor (αvβ3) Antagonist With Efficacy in a Restenosis Model,” Bioorg.Med. Chem. Lett. 8: 3171-3176 (1998). Patents assigned to SmithKlineBeecham that disclose such benzazepine, as well as relatedbenzodiazepine and benzocycloheptene, αvβ3 integrin receptor antagonistsinclude WO 96/00574, WO 96/00730, WO 96/06087, WO 96/26190, WO 97/24119,WO 97/24122, WO 97/24124, WO 98/14192, WO 98/15278, WO 99/05107, WO99/06049, WO 99/15170, WO 99/15178, WO 99/15506, and U.S. Pat. No.6,159,964, and to Genentech include WO 97/34865. Thedibenzocycloheptene, dibenzocycloheptane and dibenzoxazepine scaffoldshave also been employed as a Gly-Asp mimetic to afford αvβ3 antagonists(see WO 97/01540, WO 98/30542, WO 99/11626, WO 99/15508, WO 00/33838,U.S. Pat. No. 6,008,213, and 6,069,158, all assigned to SmithKlineBeecham).

[0015] Other integrin receptor antagonists incorporating backboneconformational ring constraints have been described in the patentliterature. Published patent applications or issued patents disclosingantagonists having a phenyl constraint include WO 98/00395, WO 99/32457,WO 99/37621, WO 99/44994, WO 99/45927, WO 99/52872, WO 99/52879, WO99/52896, WO 00/06169, EP 0 820,988, EP 0 820,991, U.S. Pat. Nos.5,741,796; 5,773,644; 5,773,646; 5,843,906; 5,852,210; 5,929,120;5,952,381; 6,028,223; and 6,040,311. Published patent applications orissued patents disclosing antagonists having a monocyclic ringconstraint include WO 99/26945, WO 99/30709, WO 99/30713, WO 99/31099,WO 99/59992, WO 00/00486, WO 00/09503, EP 0 796,855, EP 0 928,790, EP 0928,793, U.S. Pat. Nos. 5,710,159; 5,723,480; 5,981,546; 6,017,926; and6,066,648. Published patent applications or issued patents disclosingantagonists having a bicyclic ring constraint include WO 98/23608, WO98/35949, WO 99/33798, EP 0 853,084, U.S. Pat. Nos. 5,760,028;5,919,792; and 5,925,655.

[0016] Reference is also made to the following reviews for additionalscientific and patent literature that concern alpha v integrinantagonists: M. E. Duggan, et al., “Ligands to the integrin receptorα_(v)β₃ , Exp. Opin. Ther. Patents, 10: 1367-1383 (2000); M. Gowen, etal., “Emerging therapies for osteoporosis,” Emerging Drugs, 5: 1-43(2000); J. S. Kerr, et al., “Small molecule α_(v) integrin antagonists:novel anticancer agents,” Exp. Opin. Invest. Drugs, 9: 1271-1291 (2000);and W. H. Miller, et al., “Identification and in vivo efficacy ofsmall-molecule antagonists of integrin α_(v)β₃ (the vitronectinreceptor),” Drug Discovery Today, 5: 397408 (2000).

[0017] However, there still remains a need for small-molecule,non-peptidic selective αv integrin receptor antagonists that displayimproved potency, pharmacodynamic, and pharmacokinetic properties, suchas oral bioavailability and duration of action, over already describedcompounds. Such compounds would provide an enhancement in the treatment,prevention, or suppression of various pathologies enumerated above thatare mediated by αv integrin receptor binding and cell adhesion andactivation.

[0018] In WO 00/48603 (Aug. 24, 2000) and WO 00/46215 (Aug. 10, 2000),we disclosed a series of dibenzazepine and benzazepine derivatives,respectively, which are αv integrin receptor antagonists. In the presentinvention, we describe novel benzazepinone derivatives with potent αvintegrin receptor antagonistic properties.

[0019] It is therefore an object of the present invention to providenovel benzazepinone derivatives, which are useful as αv integrinreceptor antagonists.

[0020] It is another object of the present invention to provide novelbenzazepinone derivatives which are useful as αvβ3 receptor antagonists.

[0021] It is another object of the present invention to provide novelbenzazepinone derivatives which are useful as αvβ5 receptor antagonists.

[0022] It is another object of the present invention to provide novelbenzazepinone derivatives which are useful as dual αvβ3/αvβ5 receptorantagonists.

[0023] It is another object of the present invention to providepharmaceutical compositions comprising the αv integrin receptorantagonists.

[0024] It is another object of the present invention to provide methodsfor making the pharmaceutical compositions of the present invention.

[0025] It is another object of the present invention to provide methodsfor eliciting an αv integrin receptor antagonizing effect in a mammal inneed thereof by administering the compounds and pharmaceuticalcompositions containing the compounds of the present invention.

[0026] It is another object of the present invention to providecompounds and pharmaceutical compositions containing the compoundsuseful for inhibiting bone resorption, restenosis, atherosclerosis,inflammatory arthritis, diabetic retinopathy, macular degeneration,angiogenesis, cancer, and metastatic tumor growth.

[0027] It is another object of the present invention to providecompounds and pharmaceutical compositions containing the compoundsuseful for treating osteoporosis.

[0028] It is another object of the present invention to provide methodsfor inhibiting bone resorption, restenosis, atherosclerosis,inflammatory arthritis, diabetic retinopathy, macular degeneration,angiogenesis, cancer, and metastatic tumor growth.

[0029] It is another object of the present invention to provide methodsfor treating osteoporosis.

[0030] These and other objects will become readily apparent from thedetailed description which follows.

SUMMARY OF THE INVENTION

[0031] The present invention relates to compounds of structural formulaI:

[0032] or a pharmaceutically acceptable salt or ester thereof.

[0033] The present invention also relates to pharmaceutical compositionscomprising the compounds of the present invention and a pharmaceuticallyacceptable carrier.

[0034] The present invention also relates to methods for making thepharmaceutical compositions containing the compounds of the presentinvention.

[0035] The present invention also relates to methods for eliciting an αvintegrin receptor antagonizing effect in a mammal in need thereof byadministering the compounds and pharmaceutical compositions containingthe compounds of the present invention.

[0036] The present invention also relates to methods for inhibiting boneresorption, restenosis, atherosclerosis, inflammatory arthritis,diabetic retinopathy, macular degeneration, angiogenesis, cancer, andmetastatic tumor growth by administering the compounds andpharmaceutical compositions containing the compound of the presentinvention.

[0037] The present invention also relates to methods for treatingosteoporosis by administering the compounds and pharmaceuticalcompositions containing the compounds of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention comprises compounds of structural formulaI:

[0039] or a pharmaceutically acceptable salt thereof;

[0040] wherein X is selected from the group consisting of

[0041] wherein the aromatic ring carbon atoms are unsubstituted orsubstituted with one R² substituent and the non-aromatic ring carbonatoms are unsubstituted or substituted with one or two R² substituents;

[0042] R² is selected from the group consisting of hydrogen, C₁₋₄ alkyl,and C₃₋₆ cycloalkyl; or two R² substituents, when on the samenon-aromatic carbon atom, are taken together with the carbon atom towhich they are attached to form a cyclopropyl group;

[0043] R¹ is hydrogen or C₁₋₄ alkyl; and

[0044] R³ and R⁴ are each independently hydrogen or C₁₋₄ alkyl.

[0045] In certain embodiments of the present invention, the compoundscorrespond to the formula II with the designated (S)-stereochemistry atthe stereogenic carbon atom marked with an asterisk:

[0046] wherein the substituents X, R¹, R², R³, and R⁴ are as describedabove.

[0047] Illustrative but nonlimiting examples of compounds of the presentinvention that are useful as αv integrin receptor antagonists are thefollowing:

[0048] or a pharmaceutically acceptable salt thereof.

[0049] For use in medicine, the salts of the compounds of this inventionrefer to non-toxic “pharmaceutically acceptable salts.” Other salts may,however, be useful in the preparation of the compounds according to theinvention or of their pharmaceutically acceptable salts. Salts of basiccompounds encompassed within the term “pharmaceutically acceptablesalts” refer to non-toxic salts of the compound of this invention whichare generally prepared by reacting the free base with a suitable organicor inorganic acid. Representative salts of basic compounds of thepresent invention include, but are not limited to, the following:acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate,borate, bromide, camsylate, carbonate, chloride, clavulanate, citrate,dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate,hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide,isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate,mesylate, methylbromide, methylnitrate, methylsulfate, mucate,napsylate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate,pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate,polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate,tannate, tartrate, teoclate, tosylate, triethiodide and valerate.Furthermore, where the compounds of the invention carries an acidicmoiety, suitable pharmaceutically acceptable salts thereof include, butare not limited to, salts derived from inorganic bases includingaluminum, ammonium, calcium, copper, ferric, ferrous, lithium,magnesium, manganic, mangamous, potassium, sodium, zinc, and the like.Particularly preferred are the ammonium, calcium, magnesium, potassium,and sodium salts. Salts derived from pharmaceutically acceptable organicnon-toxic bases include salts of primary, secondary, and tertiaryamines, cyclic amines, and basic ion-exchange resins, such as arginine,betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol,2-methyl-2-amino-1-propanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine,tromethamine, and the like.

[0050] The compounds of the present invention can have chiral centersand can thus occur as racemates, racemic mixtures, single enantiomers,diastereomeric mixtures, and individual diastereomers, with all isomericforms being included in the present invention. Therefore, where acompound is chiral, the separate enantiomers or diastereomers,substantially free of the other, are included within the scope of theinvention; further included are all mixtures of the two enantiomers.

[0051] Some of the compounds described herein contain olefinic doublebonds, and unless specified otherwise, are meant to include both E and Zgeometric isomers.

[0052] Some of the compounds described herein may exist with differentpoints of attachment of hydrogen, referred to as tautomers. Such anexample may be a ketone and its enol form, known as keto-enol tautomers.The individual tautomers as well as mixtures thereof are encompassedwithin the compounds of the present invention.

[0053] Compounds of the present invention may be separated intodiastereoisomeric pairs of enantiomers by, for example, fractionalcrystallization from a suitable solvent, for example, methanol or ethylacetate or a mixture thereof. The pair of enantiomers thus obtained maybe separated into individual stereoisomers by conventional means, forexample, by the use of an optically active acid as a resolving agent, orby HPLC using a chiral stationary phase. Alternatively, any enantiomerof a compound of the present invention may be obtained by stereospecificsynthesis using optically pure starting materials or reagents of knownconfiguration.

[0054] Also included within the scope of the invention are polymorphsand solvates, such as hydrates, of the compounds of the instantinvention.

[0055] The present invention includes within its scope prodrugs of thecompounds of this invention. In general, such prodrugs will befunctional derivatives of the compounds of this invention which arereadily convertible in vivo into the required compounds. Thus, in themethods of treatment of the present invention, the term “administering”shall encompass the treatment of the various conditions described withthe compounds specifically disclosed or with compounds which may not bespecifically disclosed, but which convert to the specified compounds invivo after administration to the patient. Conventional procedures forthe selection and preparation of suitable prodrug derivatives aredescribed, for example, in “Design of Prodrugs,” ed. H. Bundgaard,Elsevier, 1985, which is incorporated by reference herein in itsentirety. Metabolites of these compounds include active species producedupon introduction of the compounds of this invention into the biologicalmilieu.

[0056] The term “therapeutically effective amount” shall mean thatamount of a drug or pharmaceutical agent that will elicit the biologicalor medical response of a tissue, system, animal or human that is beingsought by a researcher or clinician.

[0057] The term “αv integrin receptor antagonist,” as used herein,refers to a compound which binds to and antagonizes either the αvβ3receptor or the αvβ5 receptor, or a compound which binds to andantagonizes a combination of these receptors (for example, a dualαvβ3/αvβ5 receptor antagonist).

[0058] The term “bone resorption,” as used herein, refers to the processby which osteoclasts degrade bone.

[0059] The compounds of the present invention displays submicromolaraffinity for the αv integrin receptors, particularly the αvβ3 and αvβ5receptors. The compounds of this invention are therefore useful fortreating mammals suffering from a bone condition caused or mediated byincreased bone resorption, who are in need of such therapy.Pharmacologically effective amounts of the compound, includingpharmaceutically acceptable salts thereof, are administered to themammal, to inhibit the activity of mammalian osteoclasts.

[0060] The compounds of the present invention are administered indosages effective to antagonize the αvβ3 receptor where such treatmentis needed, as, for example, in the prevention or treatment ofosteoporosis.

[0061] Illustrating the invention is the method wherein the αv integrinreceptor antagonizing effect is an αvβ3 antagonizing effect. Moreparticularly, the αvβ3 antagonizing effect is selected from inhibitionof: bone resorption, restenosis, angiogenesis, diabetic retinopathy,macular degeneration, inflammatory arthritis, cancer, and metastatictumor growth. In one embodiment of the method, the αvβ3 antagonizingeffect is the inhibition of bone resorption.

[0062] Another example of the invention is the method wherein the αvintegrin receptor antagonizing effect is an αvβ5 antagonizing effect.More specifically, the αvβ5 antagonizing effect is selected frominhibition of restenosis, angiogenesis, diabetic retinopathy, maculardegeneration, inflammatory arthritis, cancer, and metastatic tumorgrowth.

[0063] Further illustrating the invention is the method wherein the αvintegrin receptor antagonizing effect is a dual αvβ3/αvβ5 antagonizingeffect. More particularly, the dual αvβ3/αvβ5 antagonizing effect isselected from inhibition of: bone resorption, restenosis, angiogenesis,diabetic retinopathy, macular degeneration, inflammatory arthritis,cancer, and metastatic tumor growth.

[0064] More particularly illustrating the invention is a pharmaceuticalcomposition comprising the compounds of the present invention and apharmaceutically acceptable carrier. Another example of the invention isa pharmaceutical composition made by combining the compounds describedabove and a pharmaceutically acceptable carrier. Another illustration ofthe invention is a process for making a pharmaceutical compositioncomprising combining the compounds described above and apharmaceutically acceptable carrier.

[0065] Further illustrating the invention is a method of treating and/orpreventing a condition mediated by antagonism of an αv integrin receptorin a mammal in need thereof, comprising administering to the mammal atherapeutically effective amount of the compounds described above.Preferably, the condition is selected from bone resorption,osteoporosis, restenosis, diabetic retinopathy, macular degeneration,angiogenesis, atherosclerosis, inflammatory arthritis, cancer, tumorgrowth, and metastasis. More preferably, the condition is selected fromosteoporosis and cancer. Most preferably, the condition is osteoporosis.

[0066] More specifically exemplifying the invention is a method ofeliciting an αv integrin antagonizing effect in a mammal in needthereof, comprising administering to the mammal a therapeuticallyeffective amount of the compounds or any of the pharmaceuticalcompositions described above. Preferably, the αv integrin antagonizingeffect is an αvβ3 antagonizing effect; more specifically, the αvβ3antagonizing effect is selected from inhibition of bone resorption,inhibition of restenosis, inhibition of atherosclerosis, inhibition ofangiogenesis, inhibition of diabetic retinopathy, inhibition of maculardegeneration, inhibition of inflammatory arthritis, and inhibition ofcancer or metastatic tumor growth. Most preferably, the αvβ3antagonizing effect is inhibition of bone resorption. Alternatively, theαv integrin antagonizing effect is an αvβ5 antagonizing effect or a dualαvβ3/αvβ5 antagonizing effect. Examples of αvβ5 antagonizing effects areinhibition of restenosis, atherosclerosis, angiogenesis, diabeticretinopathy, macular degeneration, inflammatory arthritis, cancer, andmetastatic tumor growth.

[0067] Additional examples of the invention are methods of inhibitingbone resorption and of treating and/or preventing osteoporosis in amammal in need thereof, comprising administering to the mammal atherapeutically effective amount of the compounds or any of thepharmaceutical compositions decribed above.

[0068] Additional illustrations of the invention are methods of treatinghypercalcemia of malignancy, osteopenia due to bone metastases,periodontal disease, hyperparathyroidism, periarticular erosions inrheumatoid arthritis, Paget's disease, immobilization-inducedosteopenia, and glucocorticoid treatment in a mammal in need thereof,comprising administering to the mammal a therapeutically effectiveamount of the compounds or any of the pharmaceutical compositionsdescribed above.

[0069] More particularly exemplifying the invention is the use of thecompounds described above in the preparation of a medicament for thetreatment and/or prevention of osteoporosis in a mammal in need thereof.Still further exemplifying the invention is the use of the compoundsdescribed above in the preparation of a medicament for the treatmentand/or prevention of bone resorption, metastatic tumor growth, cancer,restenosis, atherosclerosis, diabetic retinopathy, macular degeneration,inflammatory arthritis, and/or angiogenesis.

[0070] Also exemplifying the invention are compositions furthercomprising an active ingredient selected from the group consisting of

[0071] a) an organic bisphosphonate or a pharmaceutically acceptablesalt or ester thereof,

[0072] b) an estrogen receptor modulator,

[0073] c) an androgen receptor modulator,

[0074] d) a cytotoxic/antiproliferative agent,

[0075] e) a matrix metalloproteinase inhibitor,

[0076] f) an inhibitor of epidermal-derived, fibroblast-derived, orplatelet-derived growth factors,

[0077] g) an inhibitor of VEGF,

[0078] h) an antibody to a growth factor or to a growth factor receptor,

[0079] i) an inhibitor of Flk-1/KDR, Flt-1, Tck/Tie-2, or Tie-1,

[0080] j) a cathepsin K inhibitor,

[0081] k) a growth hormone secretagogue,

[0082] l) an inhibitor of osteoclast proton ATPase,

[0083] m) an inhibitor of urokinase plasminogen activator (u-PA),

[0084] n) a tumor-specific antibody-interleukin-2 fusion protein,

[0085] o) an inhibitor of HMG-CoA reductase,

[0086] p) a farnesyl transferase inhibitor or a geranylgeranyltransferase inhibitor or a dual farnesyl/geranylgeranyl transferaseinhibitor, and

[0087] q) a parathyroid hormone (PTH) analog;

[0088] and mixtures thereof.

[0089] (See, B. Millauer et al., “Dominant-Negative Inhibition of Flk-1Suppresses the Growth of Many Tumor Types in Vivo”, Cancer Research, 56,1615-1620 (1996), which is incorporated by reference herein in itsentirety).

[0090] Preferably, the active ingredient is selected from the groupconsisting of:

[0091] a) an organic bisphosphonate or a pharmaceutically acceptablesalt or ester thereof,

[0092] b) an estrogen receptor modulator,

[0093] c) an androgen receptor modulator,

[0094] d) an inhibitor of osteoclast proton ATPase,

[0095] e) a parathyroid hormone (PTH) analog, and

[0096] f) a cathepsin K inhibitor; and mixtures thereof.

[0097] Nonlimiting examples of such bisphosphonates include alendronate,cimadronate, clodronate, etidronate, ibandronate, pamidronate,piridronate, risedronate, tiludronate, and zolendronate, andpharmaceutically acceptable salts and esters thereof. A particularlypreferred bisphosphonate is alendronate, especially alendronatemonosodium trihydrate.

[0098] Nonlimiting examples of estrogen receptor modulators includeestrogen, progesterin, estradiol, droloxifene, raloxifene, andtamoxifene.

[0099] Nonlimiting examples of cytotoxic/antiproliferative agents aretaxol, vincristine, vinblastine, and doxorubicin.

[0100] Cathepsin K, formerly known as cathepsin O2, is a cysteineprotease and is described in PCT International Application PublicationNo. WO 96/13523, published May 9, 1996; U.S. Pat. No. 5,501,969, issuedMar. 3, 1996; and U.S. Pat. No. 5,736,357, issued Apr. 7, 1998, all ofwhich are incorporated by reference herein in their entirety. Cysteineproteases, specifically cathepsins, are linked to a number of diseaseconditions, such as tumor metastasis, inflammation, arthritis, and boneremodeling. At acidic pH's, cathepsins can degrade type-I collagen.Cathepsin protease inhibitors can inhibit osteoclastic bone resorptionby inhibiting the degradation of collagen fibers and are thus useful inthe treatment of bone resorption diseases, such as osteoporosis.

[0101] Members of the class of HMG-CoA reductase inhibitors, known asthe “statins,” have been found to trigger the growth of new bone,replacing bone mass lost as a result of osteoporosis (see The WallStreet Journal, Friday, Dec. 3, 1999, page B1). Therefore, the statinshold promise for the treatment of bone resorption. Nonlimiting examplesof statins are lovastatin, simvastatin, atorvastatin, pravastatin,fluvastatin, cerivastatin, and rosuvastatin.

[0102] Evidence for crucial role of the urokinase-urokinase receptor(u-PA-u-PAR) in angiogenesis, tumor invasion, inflammation, and matrixremodeling during wound healing and development has been presented [seeY. Koshelnick et al., “Mechanisms of signaling through UrokinaseReceptor and the Cellular Response,” Thrombosis and Haemostasis 82:305-311 (1999) and F. Blasi, “Proteolysis, Cell Adhesion, Chemotaxis,and Invasiveness Are Regulated by the u-PA-u-PAR-PAI-1 System,”Thrombosis and Haemostasis 82: 298-304 (1999)]. Thus, specificantagonists of the binding of u-PA to u-PAR inhibit cell-surfaceplasminogen activation, tumor growth, and angiogenesis in both in vitroand in vivo models.

[0103] H. N. Lode and coworkers in PNAS USA 96: 1591-1596 (1999) haveobserved synergistic effects between an antiangiogenic αv integrinantagonist and a tumor-specific antibody-cytokine (interleukin-2) fusionprotein in the eradication of spontaneous tumor metastases. Theirresults suggested this combination as having potential for the treatmentof cancer and metastatic tumor growth.

[0104] The proton ATPase which is found on the apical membrane of theosteoclast has been reported to play a significant role in the boneresorption process. Therefore, this proton pump represents an attractivetarget for the design of inhibitors of bone resorption which arepotentially useful for the treatment and prevention of osteoporosis andrelated metabolic diseases (see C. Farina et al., “Selective inhibitorsof the osteoclast vacuolar proton ATPase as novel bone antiresorptiveagents,” DDT, 4: 163-172 (1999)).

[0105] Evidence has been presented that androgenic steroids play aphysiological role in the development of bone mass in men and women andthat androgens act directly on bone. Androgen receptors have beendemonstrated in human osteoblast-like cell lines and androgens have beenshown to directly stimulate bone cell proliferation and differentiation.For a discussion, reference is made to S. R. Davis, “The therapeutic useof androgens in women,” J. Steroid Biochem. Mol. Biol., 69: 177-184(1999) and K. A. Hansen and S. P. T. Tho, “Androgens and Bone Health,”Seminars in Reproductive Endocrinology,” 16: 129-134 (1998). Thus,androgen receptor modulators may have utility in the treatment andprevention of bone loss in women.

[0106] The angiogenic factor VEGF has been shown to stimulate thebone-resorbing activity of isolated mature rabbit osteoclasts viabinding to its receptors on osteoclasts (see M. Nakagawa et al.,“Vascular endothelial growth factor (VEGF) directly enhancesosteoclastic bone resorption and survival of mature osteoclasts,” FEBSLetters, 473: 161-164 (2000)). Therefore, the development of antagonistsof VEGF binding to osteoclast receptors, such as KDR/Flk-1 and Flt-1,may provide yet a further approach to the treatment or prevention ofbone resorption.

[0107] Activators of the peroxisome proliferator-activated receptor-γ(PPARγ), such as the thiazolidinediones (TZD's), inhibit osteoclast-likecell formation and bone resorption in vitro. Results reported by R.Okazaki et al. in Endocrinology, 140, pp 5060-5065, (1999) point to alocal mechanism on bone marrow cells as well as a systemic one onglucose metabolism. Nonlimiting examples of PPARγ activators includetroglitazone, pioglitazone, rosiglitazone, and BRL 49653.

[0108] The use of parathyroid hormone (PTH) for the treatment ofosteoporosis has been suggested in the art. PTH has been found toincrease the activity of osteoblasts, the cells that form bone, therebypromoting the synthesis of new bone (Modem Drug Discovery, Vol. 3, No.8, 2000). In studies reported at the First World Congress onOsteoporosis held in Chicago in June 2000, women in combinedPTH-estrogen therapy exhibited a 12.8% increase in spinal bone mass anda 4.4% increase in total hip mass. Another study presented at the samemeeting showed that PTH could increase bone size as well as density. Aclinical trial of the effect of the human parathyroid hormone 1-34fragment [hPTH(1-34)] on postmenopausal osteoporotic women resulted in≧65% reduction in spine fractures and a 54% reduction in nonvertebralfractures, after a median of 22 months of treatment [see J. M. Hock,Bone, 27: 467-469 (2000) and S. Mohan, et al., Bone, 27: 471-478 (2000),and references cited therein]. Thus, PTH and fragments thereof, such ashPTH(1-34), may prove to be efficacious in the treatment of osteoporosisalone or in combination with other agents, such as the αvβ3 integrinantagonists of the present invention.

[0109] The present invention is also directed to combinations of thecompounds of the present invention with one or more agents useful in theprevention or treatment of osteoporosis. For example, the compounds ofthe instant invention may be effectively administered in combinationwith effective amounts of other agents such as an organicbisphosphonate, an estrogen receptor modulator, an androgen receptormodulator, a cathepsin K inhibitor, an HMG-CoA reductase inhibitor, aPPARγ activator, a VEGF receptor antagonist, an inhibitor of theosteoclast proton ATPase, or a PTH analog.

[0110] Additional illustrations of the invention are methods of treatingcancer or metastatic tumor growth in a mammal in need thereof,comprising administering to the mammal a therapeutically effectiveamount of a compounds described above and one or more agents known to becytotoxic/antiproliferative. Also, the compounds of the presentinvention can be administered in combination with radiation therapy fortreating cancer and metastatic tumor growth.

[0111] In addition, the integrin αvβ3 antagonist of the presentinvention may be effectively administered in combination with a growthhormone secretagogue in the therapeutic or prophylactic treatment ofdisorders in calcium or phosphate metabolism and associated diseases.These diseases include conditions which can benefit from a reduction inbone resorption. A reduction in bone resorption should improve thebalance between resorption and formation, reduce bone loss or result inbone augmentation. A reduction in bone resorption can alleviate the painassociated with osteolytic lesions and reduce the incidence and/orgrowth of those lesions. These diseases include: osteoporosis (includingestrogen deficiency, immobilization, glucocorticoid-induced and senile),osteodystrophy, Paget's disease, myositis ossificans, Bechterew'sdisease, malignant hypercalcemia, metastatic bone disease, periodontaldisease, cholelithiasis, nephrolithiasis, urolithiasis, urinarycalculus, hardening of the arteries (sclerosis), arthritis, bursitis,neuritis and tetany. Increased bone resorption can be accompanied bypathologically high calcium and phosphate concentrations in the plasma,which would be alleviated by this treatment. Similarly, the presentinvention would be useful in increasing bone mass in patients withgrowth hormone deficiency. Thus, preferred combinations are simultaneousor alternating treatments of an αvβ3 receptor antagonist of the presentinvention and a growth hormone secretagogue, optionally including athird component comprising an organic bisphosphonate, preferablyalendronate monosodium trihydrate.

[0112] In accordance with the method of the present invention, theindividual components of the combination can be administered separatelyat different times during the course of therapy or concurrently individed or single combination forms. The instant invention is thereforeto be understood as embracing all such regimes of simultaneous oralternating treatment, and the term “administering” is to be interpretedaccordingly. It will be understood that the scope of combinations of thecompounds of this invention with other agents useful for treatingintegrin-mediated conditions includes in principle any combination withany pharmaceutical composition useful for treating osteoporosis.

[0113] As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

[0114] The compounds of the present invention can be administered insuch oral dosage forms as tablets, capsules (each of which includessustained release or timed release formulations), pills, powders,granules, elixirs, tinctures, suspensions, syrups and emulsions.Likewise, it may also be administered in intravenous (bolus orinfusion), intraperitoneal, topical (e.g., ocular eyedrop),subcutaneous, intramuscular or transdermal (e.g., patch) form, all usingforms well known to those of ordinary skill in the pharmaceutical arts.An effective but non-toxic amount of the compounds desired can beemployed as an (αvβ3 antagonist.

[0115] The dosage regimen utilizing the compounds of the presentinvention is selected in accordance with a variety of factors includingtype, species, age, weight, sex and medical condition of the patient;the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular compound or salt thereof employed. An ordinarily skilledphysician, veterinarian or clinician can readily determine and prescribethe effective amount of the drug required to prevent, counter or arrestthe progress of the condition.

[0116] Oral dosages of the present invention, when used for theindicated effects, will range between about 0.01 mg per kg of bodyweight per day (mg/kg/day) to about 100 mg/kg/day, preferably 0.01 to 10mg/kg/day, and most preferably 0.1 to 5.0 mg/kg/day. For oraladministration, the compositions are preferably provided in the form oftablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,25.0, 50.0, 100 and 500 milligrams of the active ingredient for thesymptomatic adjustment of the dosage to the patient to be treated. Amedicament typically contains from about 0.01 mg to about 500 mg of theactive ingredient, preferably, from about 1 mg to about 100 mg of activeingredient. Intravenously, the most preferred doses will range fromabout 0.1 to about 10 mg/kg/minute during a constant rate infusion.Advantageously, the compound of the present invention may beadministered in a single daily dose, or the total daily dosage may beadministered in divided doses of two, three or four times daily.Furthermore, the compound of the present invention can be administeredin intranasal form via topical use of suitable intranasal vehicles, orvia transdermal routes, using those forms of transdermal skin patcheswell known to those of ordinary skill in the art. To be administered inthe form of a transdermal delivery system, the dosage administrationwill, of course, be continuous rather than intermittent throughout thedosage regimen.

[0117] In the methods of the present invention, the compounds hereindescribed in detail can form the active ingredient, and is typicallyadministered in admixture with suitable pharmaceutical diluents,excipients or carriers (collectively referred to herein as ‘carrier’materials) suitably selected with respect to the intended form ofadministration, that is, oral tablets, capsules, elixirs, syrups and thelike, and consistent with conventional pharmaceutical practices.

[0118] For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic, pharmaceutically acceptable, inert carrier such as lactose,starch, sucrose, glucose, methyl cellulose, magnesium stearate,dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like;for oral administration in liquid form, the oral drug components can becombined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water and the like. Moreover, whendesired or necessary, suitable binders, lubricants, disintegratingagents and coloring agents can also be incorporated into the mixture.Suitable binders include starch, gelatin, natural sugars such as glucoseor beta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include sodium oleate, sodium stearate, magnesium stearate, sodiumbenzoate, sodium acetate, sodium chloride and the like. Disintegratorsinclude, without limitation, starch, methyl cellulose, agar, bentonite,xanthan gum and the like.

[0119] The compounds of the present invention can also be administeredin the form of liposome delivery systems, such as small unilamellarvesicles, large unilamellar vesicles and multilamellar vesicles.Liposomes can be formed from a variety of phospholipids, such ascholesterol, stearylamine or phosphatidylcholines.

[0120] The compounds of the present invention may also be delivered bythe use of monoclonal antibodies as individual carriers to which thecompound molecules are coupled. The compounds of the present inventionmay also be coupled with soluble polymers as targetable drug carriers.Such polymers can include polyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamide-phenol,polyhydroxy-ethylaspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyglycolic acid, copolymers of polylactic andpolyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates andcrosslinked or amphipathic block copolymers of hydrogels.

[0121] In the Schemes and Examples below, various reagent symbols andabbreviations have the following meanings: AcOH: Acetic acid Ar: Argon9-BBN: 9-Borabicyclo[3.3.1]nonane BOC(Boc): t-Butyloxycarbonyl CBZ(Cbz):Carbobenzyloxy or benzyloxycarbonyl CH₂Cl₂: Methylene chloride CH₃CN:Acetonitrile CHCl₃: Chloroform DIAD: Diisopropyl azodicarboxylate DIPEA:Diisopropylethylamine DMAP: 4-Dimethylaminopyridine DMF:N,N-Dimethylformamide DMSO: Dimethylsulfoxide DPPF:1,1′-Bis(diphenylphosphino)ferrocene. EtOAc: Ethyl acetate EtOH: EthanolHPLC: High-performance liquid chromatography K₂CO₃: Potassium carbonateLDA: Lithium diisopropylamide MeOH: Methanol MgSO₄: Magnesium sulfateNEt₃: Triethylamine NMM: N-methylmorpholine Pd/C: Palladium on activatedcarbon catalyst PPh₃: Triphenylphosphine Ph: Phenyl RT: Room temperaturepTSA p-Toluenesulfonic acid TEA: Triethylamine TFA: Trifluoroacetic acidTHF: Tetrahydrofuran TLC: Thin Layer Chromatography

[0122] The compounds of structural formula I can be prepared by theprocedures detailed in Schemes and the Examples below. Those skilled inthe art will readily understand that known variations of the conditionsand processes of the following preparative procedures can be used toprepare the compounds. Unless stated otherwise, all operations werecarried out at room or ambient temperature, and all temperatures aredegrees Celsius.

[0123] Step A: Methyl(4S)-3-oxo-8-[2-(5,6,7,8-tetrahydro-1,8-nalphthyridin-2-yl)ethoxy]-2-(2,2,2-trifluoroethyl)-2,3,4,5-tetrahydro-1H-2-benzazepin-4-acetate(1-3)

[0124] A solution of 1-1 (0.216 g, 1.21 mmol; prepared as described inWO 99/45927), 1-2 (0.40 g, 1.21 mmol; prepared as described in WO98/14192), and triphenylphosphine (0.38 g, 1.45 mmol) in 9:1 THF/DMF (5mL) was cooled to 0° C., and diisopropyl azodicarboxylate (DIAD) (0.285mL, 1.45 mmol) was slowly added. The mixture was allowed to warm toambient temperature and stir for 15 hours, then concentrated. Silica gelchromatography (0-5% MeOH/EtOAc) provided 1-3 as a colorless oil (70mg).

[0125] Mass spectrum: Calculated for C₂₅H₂₈F₃N₃O₄: 491.2; found: 492.1(M+H).

[0126] Step B:(4S)-3-Oxo-8-[2-(5.6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy]-2-(2,2,2-trifluoroethyl)-2.3.4.5-tetrahydro-1H-2-benzazepin-4-aceticacid (1-4)

[0127] To a solution of 1-3 in dioxane (2 mL) was added NaOH (0.60 mL of1.0 N in water). After 3 hours, an additional 0.6 mL of the NaOHsolution was added. After a further 6 hours, HCl (1.2 mL 1.0 N in water)was added, and the mixture concentrated to dryness. Silica gelchromatography (20:10:1:1 to 10:10:1:1 EtOAc/EtOH/H₂O/NH₄OH) provided1-4 as a colorless solid (62 mg).

[0128] Mass spectrum: Calculated for C₂₄H₂₆F₃N₃O₄: 477.2; found: 478.1(M+H).

[0129]¹H NMR (CD₃OD): δ 7.31 (1H, d, J=7.3 Hz), 7.01 (1H, d, J=8.5 Hz),6.78 (1H, d, J=8.5 Hz), 6.72 (1H, s), 6.55 (1H, d, J=7.3 Hz), 5.32 (1H,d, J=16.6 Hz), 4.29-4.21 (3H, m), 4.32-4.05 (2H, m), 3.81 (1H, m), 3.42(2H, m), 3.06-2.99 (3H, m), 2.86-2.78 (1H, m), 2.76-2.72 (3H, m), 2.42(1H, m), 1.89 (2H, m).

EXAMPLE 2

[0130]

[0131] 6-(Methylpyridin-3-yl) trifluoromethanesulfonate (2-2)

[0132] To a suspension of 2-methyl-5-hydroxypyridine (2-1, 5 g, 46 mmol)in THF (50 mL) at −78° C. was added dropwise n-butyllithium (18.3 mL of2.5 M in hexanes, 46 mmol), producing a clear solution.Trifluoromethanesulfonic anhydride (7.69 mL, 46 mmol) was then addeddropwise, producing a deep red-colored solution. After 0.25 hours, thereaction was quenched by the addition of 100 mL of 1N NaOH. The mixturewas diluted with ether, separated, and the organics washed with water,then dried over magnesium sulfate. Evaporation of the solvents produced2-2 as a red oil (6.1 g).

[0133]¹H NMR (CDCl₃): δ 8.48 (s, 1H), 7.56 (m, 1H), 7.24 (m, 1H), 2.62(s, 3H).

[0134] 2-(But-3-enyl)-isoindole-1,3-dione (2-3)

[0135] To a stirred solution of 4-bromo-1-butene (20 g, 148 mmol) in DMF(150 mL) was added potassium phthalimide (25 g, 133 mmol) and themixture stirred for 18 hours at 70° C. After cooling to RT, the mixturewas diluted with ether, washed with water and brine, dried over MgSO₄,and concentrated to give 2-3 as a white solid.

[0136]¹H NMR (400 MHz, CDCl₃): δ 7.85 (m, 2H), 7.72 (m, 2H), 5.82 (m,1H), 5.08 (m, 2H), 3.77 (t, 2H, J=7 Hz), 2.44 (m, 2H).

[0137] 2-[4-(6-Methylpyridin-3-yl)butyl]-1H-isoindole-1,3(2H)-dione(2-4)

[0138] To a suspension of 2-3 (12.7 g, 63.2 mmol) was added a solutionof 9-BBN in THF (132 mL of a 0.5 M solution, 65.8 mmol). After stirringfor 15 hours, potassium carbonate powder (14.5 g, 105 mmol) and 2-3(12.7 g, 52.7 mmol) was added. To this mixture was then added apremixed, degassed, aged (80° C. for 30 minutes with vigorous stirring)suspension of Pd(OAc)₂ (1.78 g, 7.9 mmol) and DPPF (4.96 g, 8.9 mmol) inDMF (50 mL) via a cannula, and the resulting mixture degassed with argonbubbling for 30 minutes. The mixture was then heated at 70° C. for 3hours. Following cooling, the mixture was reduced to a thick slurry,diluted with ether, washed with water and brine. The organics were driedover MgSO₄ and concentrated to give a brown oil. This residue waschromatographed on silica gel (40-60% EtOAc/hexanes) to give 2-4 (9.6 g)as a yellow solid.

[0139]¹H NMR (400 MHz, CDCl₃): δ 8.32 (s, 1H), 7.83 (d, 2H), 7.73 (d,2H), 7.38 (m, 1H), 7.02 (d, 1H), 3.70 (t, 2H), 2.62 (t, 2H), 2.43 (s,3H), 1.74 (m, 2H), 1.66 (m, 2H).

[0140] 4-(6-Methylpyridin-3-yl)butan-1-amine (2-5)

[0141] A mixture of 2-4 (7.2 g, 24.5 mmol), hydrazine (3.8 mL), andethanol (500 mL) was heated at reflux for 4 hours. The resulting mixturewas cooled and filtered, and the filtrate concentrated to give 2-5 as ayellow solid (4.7 g) contaminated with some phthalhydrazide.

[0142]¹H NMR (400 MHz, CDCl₃): δ 8.30 (s, 1H), 7.37 (m, 1H), 7.03 (m,1H), 2.82 (br s, 2H), 2.78 (t, 2H), 2.58 (t, 2H), 2.52 (s, 3H), 1.68 (m,2H), 1.54 (m, 2H).

[0143] 2-Methyl-6.7,8,9-tetrahydro-5H-pyido[2.3-b]azepine (2-6)

[0144] A mixture of 2-5 (4.02 g, 24.5 mmol), sodium hydride (2.93 g of60% in mineral oil, 73.4 mmol), and xylenes (100 mL) was stirred for 30minutes and purged with argon, then heated at 130° C. for 4 days. Themixture was quenched with 10% potassium carbonate, diluted with ethylacetate, and washed with brine. The organics were dried over MgSO₄ andconcentrated. This residue was chromatographed on silica gel (0-100%EtOAc/hexanes) to give 2-6 (2.2 g) as a yellow solid.

[0145]¹H NMR (400 MHz, CDCl₃): δ 8.32 (s, 1H), 7.83 (d, 2H), 7.73 (d,2H), 7.38 (m, 1H), 7.02 (d, 1H), 3.70 (t, 2H), 2.62 (t, 2H), 2.43 (s,3H), 1.74 (m, 2H), 1.66 (m, 2H). Utilizing the procedures for thepreparation of 1-1 disclosed in WO 99/45927, 2-6 was then converted to2-7. Then 2-7 was converted to 2-8 using the method for the preparationof 1-4 from 1-1 described in Scheme 1.

[0146][(4S)-3-Oxo-8-[2-(6,7,8,9-tetrahydro-5H-pyrido[2,3-b]azepin-2-yl)ethoxy]-2-(2,2,2-trifluoroethyl)-2,3,4,5-tetrahydro-1H-2-benzazepin-4-yl]aceticacid (2-8)

[0147]¹H NMR (CD₃OD): δ 7.38 (d, 1H), 7.02 (d, 1H), 6.78 (d, 1H), 6.72(m, 2H), 5.32 (d, 1H), 4.26 (m, 3H), 4.12 (m, 2H), 3.81 (s, 1H), 3.19(m, 2H), 3.06 (m, 3H), 2.88-2.71 (m, 4H), 2.48 (s, 1H), 1.84 (m, 2H),1.75 (m, 2H). Mass spectrum: Calculated for C₂₄H₂₆F₃N₃O₄: 491.5; found:492.1 (M+H).

EXAMPLE 3

[0148]

[0149] EXAMPLE 3 (compound 3-13) is prepared as outlined in SCHEME 3.

EXAMPLE 4

[0150]

[0151] EXAMPLE 4 (compound 4-7) is prepared as outlined in SCHEME 4.

[0152] N-(4-Iodo-phenylsulfonylamino)-L-asparagine (A-2)

[0153] To a stirred solution of acid A-1 (4.39 g, 33.2 mmol), NaOH (1.49g, 37.2 mmol), dioxane (30 ml) and H₂O (30 ml) at 0° C. was added pipsylchloride (10.34 g, 34.2 mmol). After ˜5 minutes, NaOH (1.49, 37.2 mmol)dissolved in 15 ml H₂O, was added followed by the removal of the coolingbath. After 2.0 h, the reaction mixture was concentrated. The residuewas dissolved in H₂O (300 ml) and then washed with EtOAc. The aqueousportion was cooled to 0° C. and then acidified with concentrated HCl.The solid was collected and then washed with Et₂O to provide acid A-2 asa white solid.

[0154]¹H NMR (300 MHz, D₂O) δ 7.86 (d, 2H, J=8 Hz), 7.48 (d, 2H, J=8 Hz)3.70 (m, 1H), 2.39 (m, 2H).

[0155] 2(S)-(4-Iodo-phenylsulfonylamino)-β-alanine (A-3)

[0156] To a stirred solution of NaOH (7.14 g, 181.8 mmol) and H₂O (40ml) at 0° C. was added Br₂ (1.30 ml, 24.9 mmol) dropwise over a tenminute period. After ˜5 minutes, acid A-2 (9.9 g, 24.9 mmol), NaOH (2.00g, 49.8 mmol) and H₂O (35 ml) were combined, cooled to 0° C. and thenadded in a single portion to the reaction. After stirring for 20 minutesat 0° C., the reaction was heated to 90° C. for 30 minutes and thenrecooled to 0° C. The pH was adjusted to −7 by dropwise addition ofconcentrated HCl. The solid was collected, washed with EtOAc, and thendried in vacuo to provide acid A-3 as a white solid.

[0157]¹H NMR (300 MHz, D₂O) δ 8.02 (d, 2H, J=8 Hz), 7.63 (d, 2H, J=8Hz), 4.36 (m, 1H), 3.51 (dd, 1H, J=5 Hz, 13 Hz) 3.21 (m, 1H).

[0158] Ethyl 2(S)-(4-iodo-phenylsulfonylamino)-β-alanine-hydrochloride(A-4)

[0159] HCl gas was rapidly bubbled through a suspension of acid A-3 (4.0g, 10.81 mmol) in EtOH (50 ml) at 0° C. for 10 minutes. The cooling bathwas removed and the reaction was heated to 60° C. After 18 h, thereaction was concentrated to provide ester A-4 as a white solid.

[0160]¹H NMR (300 MHz, CD₃OD) δ 7.98 (d, 2H, J=8 Hz), 7.63 (d, 2H, J=8Hz), 4.25 (q, 1H, J=5 Hz), 3.92 (m, 2H), 3.33 (m, 1H), 3.06 (m, 1H),1.01 (t, 3H, J=7 Hz).

[0161] Ethyl 4-[2-(2-Aminopyridin-6-yl)ethyl]benzoate (A-5a)

[0162] A mixture of ester A-5 (700 mg, 2.63 mmol), (for preparation,see: Scheme 29 (intermediate 29-3) of U.S. Pat. No. 5,741,796 (Apr. 21,1998)), 10% Pd/C (350 mg) and EtOH were stirred under 1 atm H₂. After 20h, the reaction was filtered through a celite pad and then concentratedto provide ester A-5a as a brown oil.

[0163] TLC R_(f)=0.23 (silica, 40% EtOAc/hexanes)

[0164]¹H NMR (300 MHz, CDCl₃) δ 7.95 (d, 2H, J=8 Hz), 7.26 (m, 3H), 6.43(d, 1H, J=7 Hz), 6.35 (d, 1H, J=8 Hz), 4.37 (m, 4H), 3.05 (m, 2H), 2.91(m, 2H), 1.39 (t, 3H, J=7 Hz).

[0165] 4-[2-(2-Aminopyridin-6-yl)ethyl]benzoic acid hydrochloride (A-6)

[0166] A suspension of ester A-5a (625 mg, 2.31 mmol) in 6N HCl (12 ml)was heated to 60° C. After ˜20 h, the reaction was concentrated to giveacid A-6 as a tan solid.

[0167]¹H NMR (300 MHz, CD₃OD) δ 7.96 (d, 2H, J=8 Hz), 7.80 (m, 1H), 7.33(d, 2H, J=8 Hz), 6.84 (d, 1H, J=9 Hz), 6.69 (d, 1H, J=7 Hz), 3.09 (m,4H).

[0168] Ethyl4-[2-(2-Aminopydin-6-yl)ethyl]benzoyl-2(S)-(4-iodo-phenylsulfonylamino)-D-alanine(A-7)

[0169] A solution of acid 15-6 (400 mg, 1.43 mmol), amine A-4 (686 mg,1.57 mmol), EDC (358 mg, 1.86 mmol), HOBT (252 mg, 1.86 mmol), NMM (632μl, 5.72 mmol) in DMF (10 ml) was stirred for ˜20 h. The reaction wasdiluted with EtOAc and then washed with sat. NaHCO₃, brine, dried(MgSO₄) and concentrated. Flash chromatography (silica, EtOAc then 5%isopropanol/EtOAc) provided amide A-7 as a white solid.

[0170] TLC R_(f)=0.4 (silica, 10% isopropanol/EtOAc)

[0171]¹H NMR (300 MHz, CD₃OD) δ 7.79 (d, 2H, J=9 Hz) 7.61 (d, 2H, J=8Hz), 7.52 (d, 2H, J=9 Hz), 7.29 (m, 1H), 7.27 (d, 2H, J=8 Hz), 4.20 (m,1H), 3.95 (q, 2H, J=7 Hz), 3.66 (dd, 1H, J=6 Hz, 14 Hz), 3.49 (dd, 1H,J=8 Hz, 13 Hz), 3.01 (m, 2H), 2.86 (m, 2H), 1.08 (t, 3H, J=7 Hz).

[0172]4-[2-(2-Aminopydin-6-yl)ethyl]benzoyl-2(S)-(4-iodophenyl-sulfonylamino)-β-alanine(A-8)

[0173] A solution of ester A-7 (200 mg, 0.3213 mmol) and 6N HCl (30 ml)was heated to 60° C. After ˜20 h, the reaction mixture was concentrated.Flash chromatography (silica, 20:20:1:1 EtOAc/EtOH/NH₄OH/H₂O) providedacid A-8 as a white solid.

[0174] TLC R_(f)=0.45 (silica, 20:20:1:1 EtOAc/EtOH/NH₄OH/H₂O)

[0175]¹H NMR (400 MHz, DMSO-d₆) δ 8.40 (m, 1H), 8.14 (Bs, 1H), 7.81 (d,2H, J=8 Hz), 7.62 (d, 2H, J=8 Hz), 7.48 (d, 2H, J=8 Hz), 7.27 (m, 3H),6.34 (d, 1H, J=7 Hz), 6.25 (d, 1H, J=8 Hz), 5.85 (bs, 2H), 3.89 (bs,1H), 3.35 (m, 2H), 2.97 (m, 2H), 2.79 (m, 2H).

[0176]4-[2-(2-Aminopyridin-6-yl)ethyl)benzoyl-2(S)-(4-trimethylstannyl-phenylsulfonylamino-β-alanine(A-9)

[0177] A solution of iodide A-8 (70 mg, 0.1178 mmol), [(CH₃)₃Sn]₂ (49μl, 0.2356 mmol), Pd(PPh₃)₄ (5 mg) and dioxane (7 ml) was heated to 90°C. After 2 h, the reaction was concentrated and then purified bypreparative HPLC (Delta-Pak C₁₈ 15 μM 100 A°, 40×100 mm; 95:5 then5:95H₂O/CH₃CN) to provide the trifluoroacetate salt. The salt wassuspended in H₂O (10 ml), treated with NH₄OH (5 drops) and thenlyophilized to provide amide A-9 as a white solid.

[0178]¹H NMR (400 MHz, DMSO-d₆) δ 8.40 (m, 1H), 8.18 (d, 1H, J=8 Hz),7.67 (m, 5H), 7.56 (d, 2H, J=8 Hz), 7.29 (d, 2H, J=8 Hz), 6.95-7.52 (m,2H), 6.45 (bs, 2H), 4.00 (m, 1H), 3.50 (m, 1H), 3.33 (m, 1H), 2.97 (m,2H), 2.86 (m, 2H).

[0179]4-[2-(2-Aminopyridin-6-yl)ethyl]benzoyl-2(S)-4-¹²⁵iodo-phenylsulfonylamino-β-alanine(A-10)

[0180] An iodobead (Pierce) was added to a shipping vial of 5 mCi ofNa¹²⁵I (Amersham, IMS30) and stirred for five minutes at roomtemperature. A solution of 0.1 mg of A-9 in 0.05 mL of 10% H₂SO₄/MeOHwas made and immediately added to the Na¹²⁵I/iodobead vial. Afterstirring for three minutes at room temperature, approximately 0.04-0.05mL of NH₄OH was added so the reaction mixture was at pH 6-7. The entirereaction mixture was injected onto the HPLC for purification [Vydacpeptide-protein C-18 column, 4.6×250 mm, linear gradient of 10%acetonitrile (0.1% (TFA):H₂O (0.1% TFA) to 90% acetonitrile (0.1%TFA):H₂O (0.1% TFA) over 30 minutes, 1 mUmin]. The retention time ofA-10 is 17 minutes under these conditions. Fractions containing themajority of the radioactivity were pooled, lyophilized and diluted withethanol to give approximately 1 mCi of A-10, which coeluted on HPLCanalysis with an authentic sample of A-8.

[0181]2(S)-(4-Iodo-benzenesulfonylamino)-3-{4-[2-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)-ethyl]-benzoylamino}-propionicacid ethyl ester (B-2)

[0182] A mixture of B-1 (0.23 g, 0.72 mmol; for preparation see U.S.Pat. No. 5,741,796), A-4 (0.343 g, 0.792 mmol), EDC (0.179 g, 0.93mmol), HOBT (0.126 g, 0.93 mmol), NMM (0.316 mL, 2.86 mmol) inacetonitrile (3 mL) and DMF (3 mL) was stirred for 2 hours at ambienttemperature then diluted with ethyl acetate, washed with water,saturated aqueous NaHCO₃, and brine, dried over MgSO₄, and concentrated.The residue was chromatographed on silica gel (70:25:5 CHCl₃/EtOAc/MeOH)to give B-2 as a white solid.

[0183] TLC R_(f)=0.22 (silica, 70:25:5 CHCl₃/EtOAc/MeOH).

[0184]¹H NMR (300 MHz, CDCl₃) δ 7.79 (d, 2H, J=8 Hz), 7.63 (d, 2H, J=8Hz), 7.54 (d, 2H, J=8 Hz), 7.27 (d, 2H, J=8 Hz),7.04 (d, 1H, J=7 Hz),6.60 (m, 1H), 6.29 (d, 1H, J=7 Hz), 4.83 (br s, 1H), 4.09 (m, 3H), 3.84(m, 1H), 3.68 (m, 1H), 3.42 (m, 2H), 3.01 (m, 4H), 2.86 (m, 4H), 2.69(t, 2H, J=6 Hz), 1.88 (m, 2H).

[0185]2(S)-(4-Iodo-benzenesulfonylamino)-3-{4-[2-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)-ethyl]-benzoylamino}-propionicacid (B-3)

[0186] A mixture of B-2 (0.38 g, 0.573 mmol) and 6N HCl (50 mL) wasstirred for 14 hours at 60° C. After cooling to room temperature, themixture was concentrated, and the residue chromatographed on silica gel(25:10:1:1 to 15:10:1:1 EtOAc/EtOH/NH₄OH/H₂O) to give B-3 as a whitesolid.

[0187] TLC R_(f)=0.43 (silica, 10:10:1:1 EtOAc/EtOH/NH₄OH/H₂O).

[0188]¹H NMR (300 MHz, DMSO-d₆) δ 8.42 (m, 1H), 7.79 (d, 2H, J=8 Hz),7.63 (d, 2H, J=8 Hz), 7.44 (d, 2H, J=8 Hz), 7.27 (d, 2H, J=8 Hz),7.10(d, 1H, J=7 Hz), 6.58 (br s, 1H), 6.32 (d, 1H, J=7 Hz), 3.96 (m, 1H),3.51 (m, 1H), 3.30 (m, 5H), 2.96 (m, 2H), 2.78 (m, 2H), 2.62 (m, 2H),1.77 (m, 2H).

[0189] HRMS: For C₂₆H₂₇N₄O₅S, expected 635.0818, found 635.0831.

[0190]3-4-[2-(5,6,7,8-Tetrahydro-[1,8]naphthyridin-2-yl)-ethyl]-benzoylamino]-2(S)-(4-trimethylstannanyl-benzenesulfonylamino)-propionicacid (B4)

[0191] A mixture of B-3 (0.10 g, 0.16 mmol), hexamethyldistannane (0.065mL, 0.32 mmol), Pd(PPh₃)₄, and dioxane (10 mL) was stirred for one hourat 90° C. After cooling to room temperature, the mixture wasconcentrated, and the residue chromatographed on silica gel (50:10:1:1to 25:10:1:1 EtOAc/EtOH(NH₄H/H₂O) to give B-4 as a white solid.

[0192] TLC R_(f)=0.48 (silica, 15:10:1:1 EtOAc/EtOH/NH₄OH/H₂O).

[0193]¹H NMR (300 MHz, DMSO-d₆) δ 8.38 (m, 1H), 8.14 (m, 1H), 7.63 (m,4H), 7.28 (d, 2H, J=8 Hz), 7.08 (d, 1H, J=7 Hz), 6.50 (br s, 1H), 6.28(d, 1H, J=7 Hz), 3.96 (m, 1H), 3.48 (m, 1H), 3.31 (m, 5H), 2.96 (m, 2H),2.78 (m, 2H), 2.62 (m, 2H), 1.77 (m, 2H), 0.28 (s, 9H).

[0194] High resolution mass spectrum: For C₂₉H₃₆N₄O₅SSn, expected665.1533 (¹¹²Sn) and 673.1507 (¹²⁰Sn), found 665.1510 and 673.1505.

[0195]2(S)-(4-¹²⁵Iodo-benzenesulfonylamino)-3-{4-[2-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)-ethyl]-benzoylamino}-propionicacid (B-5)

[0196] A stir bar, methanol (0.05 mL) and an iodobead (Pierce) wereadded to a shipping vial of Na¹²⁵, (10 mCi, Amersham, IMS300) andstirred for five minutes at room temperature. A solution of B4 (−0.1 mg)in methanol (0.04 mL) was made and a portion (0.02 mL) was added to amixture of H₂SO₄ (0.005 mL) in methanol (0.025 mL), and this solutionwas added immediately to the Na¹²⁵I/iodobead vial. After stirring fortwo minutes at room temperature, the reaction was quenched with NH₄OH(0.04-0.05 mL) and the entire reaction mixture was injected onto theHPLC for purification [Vydac peptide-protein C-18 column, 4.6×250 mm,linear gradient of 10% acetonitrile:H₂O (0.1% TFA) to 90%acetonitrile:H₂O (0.1% TFA) over 20 minutes, 1 mLmin]. The retentiontime of B-5 is 16 minutes under these conditions. Fractions containingthe majority of the radioactivity were pooled, lyophilized and dilutedwith ethanol to give approximately 1 mCi of B-5, which coeluted on HPLCanalysis with an authentic sample of B-3.

[0197] Instrumentation: Analytical and preparative HPLC was carried outusing a Waters 600E Powerline Multi Solvent Delivery System with 0.1 mLheads with a Rheodyne 7125 injector and a Waters 990 Photodiode ArrayDetector with a Gilson FC203 Microfraction collector. For analytical andpreparative HPLC, a Vydac peptide-protein C-18 column, 4.6×250 mm wasused with a C-18 Brownlee modular guard column. The acetonitrile usedfor the HPLC analyses was Fisher Optima grade. The HPLC radiodetectorused was a Beckman 170 Radioisotope detector. A Vydac C-18 protein andpeptide column, 3.9×250 mm was used for analytical and preparative HPLC.Solutions of radioactivity were concentrated using a Speedvac vacuumcentrifuge. Calibration curves and chemical concentrations weredetermined using a Hewlett Packard Model 8452A UV/Vis Diode ArraySpectrophotometer. Sample radioactivities were determined in a PackardA5530 gamma counter.

[0198] The test procedures employed to measure αvβ3 and αvβ5 binding andthe bone resorption inhibiting activity of the compound of the presentinvention are described below.

[0199] Bone Resorption-Pit Assay

[0200] When osteoclasts engage in bone resorption, they can cause theformation of pits in the surface of bone that they are acting upon.Therefore, when testing compounds for their ability to inhibitosteoclasts, it is useful to measure the ability of osteoclasts toexcavate these resorption pits when the inhibiting compound is present.

[0201] Consecutive 200 micron thick cross sections from a 6 mm cylinderof bovine femur diaphysis are cut with a low speed diamond saw (Isomet,Beuler, Ltd., Lake Bluff, E). Bone slices are pooled, placed in a 10%ethanol solution and refrigerated until further use.

[0202] Prior to experimentation, bovine bone slices are ultrasonicatedtwice, 20 minutes each in H₂O. Cleaned slices are placed in 96 wellplates such that two control lanes and one lane for each drug dosage areavailable. Each lane represents either triplicate or quadruplicatecultures. The bone slices in 96 well plates are sterilized by UVirradiation. Prior to incubation with osteoclasts, the bone slices arehydrated by the addition of 0.1 ml αMEM, pH 6.9 containing 5% fetalbovine serum and 1% penicillin/streptomycin.

[0203] Long bones from 7-14 day old rabbits (New Zealand White Hare) aredissected, cleaned of soft tissue and placed in αMEM containing 20 mMHEPES. The bones are minced using scissors until the pieces are <1 mmand transferred to a 50 ml tube in a volume of 25 ml. The tube is rockedgently by hand for 60 cycles, the tissue is sedimented for 1 min., andthe supernatant is removed. Another 25 ml of medium is added to thetissue and rocked again. The second supernatant is combined with thefirst. The number of cells is counted excluding erythrocytes (typically˜2×10⁷ cells/ml). A cell suspension consisting of 5×10⁶/ml in αMEMcontaining 5% fetal bovine serum, 10 nM 1,25(OH)₂D₃, andpencillin-streptomycin is prepared. 200 ml aliquots are added to bovinebone slices (200 mm×6 mm) and incubated for 2 hrs. at 37° C. in ahumidified 5% CO₂ atmosphere. The medium is removed gently with amicropipettor and fresh medium containing test compounds is added. Thecultures are incubated for 48 hrs., and assayed for c-telopeptide(fragments of the al chain of type I collagen) by Crosslaps for culturemedia (Herlev, Denmark).

[0204] Bovine bone slices are exposed to osteoclasts for 20-24 hrs andare processed for staining. Tissue culture media is removed from eachbone slice. Each well is washed with 200 ml of H₂O, and the bone slicesare then fixed for 20 minutes in 2.5% glutaraldehyde, 0.1 M cacodylate,pH 7.4. After fixation, any remaining cellular debris is removed by 2min. ultrasonication in the presence of 0.25 M NH₄OH followed by 2×15min ultrasonication in H₂O. The bone slices are immediately stained for6-8 min with filtered 1% toluidine blue and 1% borax.

[0205] After the bone slices have dried, resorption pits are counted intest and control slices. Resorption pits are viewed in a Microphot Fx(Nikon) fluorescence microscope using a polarizing Nikon IGS filtercube. Test dosage results are compared with controls and resulting IC₅₀values are determined for each compound tested.

[0206] The appropriateness of extrapolating data from this assay tomammalian (including human) disease states is supported by the teachingfound in Sato, M., et al., Journal of Bone and Mineral Research, Vol. 5,No. 1, pp. 31-40, 1990, which is incorporated by reference herein in itsentirety. This article teaches that certain bisphosphonates have beenused clinically and appear to be effective in the treatment of Paget'sdisease, hypercalcemia of malignancy, osteolytic lesions produced bybone metastases, and bone loss due to immobilization or sex hormonedeficiency. These same bisphosphonates are then tested in the resorptionpit assay described above to confirm a correlation between their knownutility and positive performance in the assay.

[0207] EIB Assay

[0208] Duong et al., J. Bone Miner. Res., 8: S378 (1993), describes asystem for expressing the human integrin αvβ3. It has been suggestedthat the integrin stimulates attachment of osteoclasts to bone matrix,since antibodies against the integrin, or RGD-containing molecules, suchas echistatin (European Publication 382 451), can effectively block boneresorption.

[0209] Reaction Mixture:

[0210] 1. 175 μl TBS buffer (50 mM Tris.HCl pH 7.2, 150 mM NaCl, 1% BSA,1 mM CaCl₂, 1 mM MgCl₂).

[0211] 2. 25 ml cell extract (dilute with 100 mM octylglucoside bufferto give 2000 cpm/25 μl).

[0212] 3. ¹²⁵I-echistatin (25 μl/50,000 cpm) (see EP 382 451).

[0213] 4. 25 μl buffer (total binding) or unlabeled echistatin(non-specific binding).

[0214] The reaction mixture was then incubated for 1 h at room temp. Theunbound and the bound αvβ3 were separated by filtration using a SkatronCell Harvester. The filters (prewet in 1.5% poly-ethyleneimine for 10mins) were then washed with the wash buffer (50 mM Tris HCl, 1 mMCaCl₂/MgCl₂, pH 7.2). The filter was then counted in a gamma counter.

[0215] SPAV3 Assay

[0216] Materials:

[0217] 1. Wheat germ agglutinin Scintillation Proximity Beads (SPA):Amersham

[0218] 2. Octylglucopyranoside: Calbiochem

[0219] 3. BEPES: Calbiochem

[0220] 4. NaCl: Fisher

[0221] 5. CaCl₂: Fisher

[0222] 6. MgCl₂: SIGMA

[0223] 7. Phenylmethylsulfonylfluoride (PMSF): SIGMA

[0224] 8. Optiplate: PACKARD

[0225] 9. Compound A-10 (specific activity 500-1000 Ci/mmole)

[0226] 10. test compound

[0227] 11. Purified integrin receptor: αvβ3 was purified from 293 cellsoverexpressing αvβ3 (Duong et al., J. Bone Min. Res., 8:S378, 1993)according to Pytela (Methods in Enzymology, 144:475, 1987)

[0228] 12. Binding buffer: 50 mM HEPES, pH 7.8, 100 mM NaCl, 1 mMCa²⁺/Mg²⁺, 0.5 mM PMSF

[0229] 13. 50 mM octylglucoside in binding buffer: 50-OG buffer

[0230] Procedure:

[0231] 1. Pretreatment of SPA beads:

[0232] 500 mg of lyophilized SPA beads were first washed four times with200 ml of 50-OG buffer and once with 100 ml of binding buffer, and thenresuspended in 12.5 ml of binding buffer.

[0233] 2. Preparation of SPA beads and receptor mixture

[0234] In each assay tube, 2.5 μl (40 mg/ml) of pretreated beads weresuspended in 97.5 μl of binding buffer and 20 ml of 50-OG buffer. 5 ml(−30 ng/μl) of purified receptor was added to the beads in suspensionwith stirring at room temperature for 30 minutes. The mixture was thencentrifuged at 2,500 rpm in a Beckman GPR Benchtop centrifuge for 10minutes at 4° C. The pellets were then resuspended in 50 μl of bindingbuffer and 25 μl of 50-OG buffer.

[0235] 3. Reaction

[0236] The following were sequentially added into Optiplate incorresponding wells:

[0237] (i) Receptor/beads mixture (75 μl)

[0238] (ii) 25 μl of each of the following: compound to be tested,binding buffer for total binding or A-8 for non-specific binding (finalconcentration 1 μM)

[0239] (iii) A-10 in binding buffer (25 μl, final concentration 40 μM)

[0240] (iv) Binding buffer (125 μl)

[0241] (v) Each plate was sealed with plate sealer from PACKARD andincubated overnight with rocking at 4° C.

[0242] 4. Plates were counted using PACKARD TOPCOUNT

[0243] 5. % inhibition was calculated as follows:

[0244] A=total counts

[0245] B=nonspecific counts

[0246] C=sample counts

% inhibition=[{(A−B)−(C−B)}/(A−B)]/(A−B)×100

[0247] OCFORM Assay

[0248] Osteoblast-like cells (1.8 cells), originally derived from mousecalvaria, were plated in CORNING 24 well tissue culture plates in αMEMmedium containing ribo- and deoxyribonucleosides, 10% fetal bovine serumand penicillin-streptomycin. Cells were seeded at 40,000/well in themorning. In the afternoon, bone marrow cells were prepared from six weekold male Balb/C mice as follows:

[0249] Mice were sacrificed, tibiae removed and placed in the abovemedium. The ends were cut off and the marrow was flushed out of thecavity into a tube with a 1 mL syringe with a 27.5 gauge needle. Themarrow was suspended by pipetting up and down. The suspension was passedthrough >100 mm nylon cell strainer. The resulting suspension wascentrifuged at 350×g for seven minutes. The pellet was resuspended, anda sample was diluted in 2% acetic acid to lyse the red cells. Theremaining cells were counted in a hemacytometer. The cells were pelletedand resuspended at 1×10⁶ cells/mL. 50 μL was added to each well of 1.8cells to yield 50,000 cells/well and 1,25-dihydroxy-vitamin D₃ (D₃) wasadded to each well to a final concentration of 10 nM. The cultures wereincubated at 37° C. in a humidified, 5% CO₂ atmosphere. After 48 h, themedium was changed. 72 h after the addition of bone marrow, testcompounds were added with fresh medium containing D₃ to quadruplicatewells. Compounds were added again after 48 h with fresh mediumcontaining D₃. After an additional 48 h., the medium was removed, cellswere fixed with 10% formaldehyde in phosphate buffered saline for 10minutes at room temperature, followed by a 1-2 minute treatment withethanol:acetone (1:1) and air dried. The cells were then stained fortartrate resistant acid phosphatase as follows:

[0250] The cells were stained for 10-15 minutes at room temperature with50 mM acetate buffer, pH 5.0 containing 30 mM sodium tartrate, 0.3 mg/mLFast Red Violet LB Salt and 0.1 mg/mL Naphthol AS-MX phosphate. Afterstaining, the plates were washed extensively with deionized water andair dried. The number of multinucleated, positive staining cells wascounted in each well.

[0251] SPAV5 Assay

[0252] Materials:

[0253] 1. Wheat germ agglutinin Scintillation Proximity Beads (SPA):Amersham

[0254] 2. Octylglucopyranoside and Phorbo-12-myristate-13-acetate (PMA):Calbiochem

[0255] 3. Tris-HCl, NaCl and CaCl₂: Fisher

[0256] 4. Minimum Essential Media (MEM): Gibco/BRL

[0257] 5. Fetal bovine serum (FBS): Hyclone

[0258] 6. MgCl₂, MnCl₂, and Phenylmethylsulfonylfluoride (PMSF): SIGMA

[0259] 7. Protease inhibitor cocktail tablets: Boehringer Mannheim.

[0260] 8. Optiplate-96 wells: PACKARD

[0261] 9. B-5 was used as radiolabeled ligand (specific activity500-1000 Ci/mmole) and B-3 (2.5 μM) was used to achieve 100% inhibition.

[0262] 10. Test compound.

[0263] 11. HEK293 cells overexpressing α_(v)β₅ integrins (Simon et al.,J. Biol. Chem. 272, 29380-29389, 1997) are cultured in 150 mm dishes in10% FBS/MEM media (Gibco/BRL).

[0264] 12. Lysis buffer: 100 mM octylglucopyranoside, 50 mM Tris, pH7.5, 100 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 0.5 mM PMSF and proteaseinhibitors (1 tablet/50 ml buffer).

[0265] 13. Binding buffer: 50 mM Tris, pH 7.5, 100 mM NaCl, 1 mM CaCl₂ 1mM MgCl₂ and 1 mM MnCl₂.

[0266] 14. 50 mM octylglucopyranoside in binding buffer: 50-OG buffer

[0267] Procedure:

[0268] 1. α_(v)β₅-cell lysates: HEK 293 cells expressing α_(v)β₅integrins were cultured until confluent. Cells were then starvedovernight in media containing 0.5% FBS, followed by treatment with 100nM PMA for 20 min. Cells were washed 2 times with cold phosphate buffersaline (4° C.) and solubilized in lysis buffer for 30 min on ice.Lysates were clarified using a Beckman JA-20 at 20,000×g. Proteinconcentration of clarified lysates was determined using a micro BCA kit(Pierce) and stored in aliquots at 80° C.

[0269] 2. Pretreatment of SPA beads:

[0270] 500 mg of lyophilized SPA beads were first washed four times with200 ml of 50-OG buffer and once with 100 ml of binding buffer, and thenresuspended in 12.5 ml of binding buffer.

[0271] 3. Preparation of SPAV5 binding reaction

[0272] To each assay well, the following were sequentially added intoOptiplate plates:

[0273] (i) Binding buffer to make up final volume of 125 μl per well.

[0274] (ii) 3 μl (120 μg/well) of pretreated beads diluted with 22 μl of50-OG Buffer

[0275] (iii) 15 μg of α_(v)β₅-cell lysate proteins.

[0276] (iv) B-5 at 50,000 cpm.

[0277] (v) 25 μl of graded concentrations of test compound.

[0278] (vi) Each plate was sealed with plate sealer from PACKARD andincubated overnight with rocking at 4° C.

[0279] 4. Plates were counted using PACKARD TOPCOUNT microplatescintillation counter.

[0280] 5. % Inhibition was calculated as follows:

[0281] A=total counts (binding of receptor to B-5)

[0282] B=nonspecific counts (binding of receptor to B-5 in the presenceof 2.5 μM cold ligand)

[0283] C=counts from receptor binding to test compound

% inhibition=[{(A−B)−(C−B)}/(A−B)]/(A−B)×100

[0284] IC₅₀ of test compound was calculated as 50% of inhibition.

Example of a Pharmaceutical Formulation

[0285] As a specific embodiment of an oral composition, 100 milligramsof a compound of the present invention are formulated with sufficientfinely divided lactose to provide a total amount of 580 to 590 mg tofill a size 0 hard gel capsule.

[0286] While the invention has been described and illustrated inreference to certain preferred embodiments thereof, those skilled in theart will appreciate that various changes, modifications andsubstitutions can be made therein without departing from the spirit andscope of the invention. For example, effective dosages other than thepreferred doses as set forth hereinabove may be applicable as aconsequence of variations in the responsiveness of the mammal beingtreated for severity of bone disorders caused by resorption, or forother indications for the compounds of the invention indicated above.Likewise, the specific pharmacological responses observed may varyaccording to and depending upon the particular active compound selectedor whether there are present pharmaceutical carriers, as well as thetype of formulation and mode of administration employed, and suchexpected variations or differences in the results are contemplated inaccordance with the objects and practices of the present invention. Itis intended, therefore, that the invention be limited only by the scopeof the claims which follow and that such claims be interpreted asbroadly as is reasonable.

What is claimed is:
 1. A compound of the structural formula I:

wherein X is selected from the group consisting of

or a pharmaceutically acceptable salt or ester thereof; wherein thearomatic ring carbon atoms are unsubstituted or substituted with one R²substituent and the non-aromatic ring carbon atoms are unsubstituted orsubstituted with one or two R² substituents; R² is selected from thegroup consisting of hydrogen, C₁₋₄ alkyl, and C₃₋₆ cycloalkyl; or two R²substituents, when on the same non-aromatic carbon atom, are takentogether with the carbon atom to which they are attached to form acyclopropyl group; R¹ is hydrogen or C₁₋₄ alkyl; and R³ and R⁴ are eachindependently hydrogen or C₁₋₄ alkyl.
 2. The compound of claim 1 whereinR¹ is hydrogen.
 3. The compound of claim 1 of structural formula IIwherein the carbon atom marked with * has the (S) configuration:


4. The compound of claim 3 which is

or a pharmaceutically acceptable salt thereof.
 5. A pharmaceuticalcomposition comprising a compound according to claim 1 and apharmaceutically acceptable carrier.
 6. The composition of claim 5 whichfurther comprises an active ingredient selected from the groupconsisting of a) an organic bisphosphonate or a pharmaceuticallyacceptable salt or ester thereof, b) an estrogen receptor modulator, c)an androgen receptor modulator, d) a cytotoxic/antiproliferative agent,e) a matrix metalloproteinase inhibitor, f) an inhibitor ofepidermal-derived, fibroblast-derived, or platelet-derived growthfactors, g) an inhibitor of VEGF, h) an antibody to a growth factor orto a growth factor receptor, i) an inhibitor of Flk-1/KDR, Flt-1,Tck/Tie-2, or Tie-2, j) a cathepsin K inhibitor, k) a growth hormonesecretagogue, l) an inhibitor of osteoclast proton ATPase, m) aninhibitor of urokinase plasminogen activator (u-PA), n) a tumor-specificantibody-interleukin-2 fusion protein, o) an inhibitor of HMG-CoAreductase, p) a farnesyl transferase inhibitor or a geranylgeranyltransferase inhibitor or a dual farnesyl/geranylgeranyl transferaseinhibitor, and q) a parathyroid hormone (PTH) analog; and mixturesthereof.
 7. The composition of claim 6 wherein said active ingredient isselected from the group consisting of a) an organic bisphosphonate or apharmaceutically acceptable salt or ester thereof, b) an estrogenreceptor modulator, c) an androgen receptor modulator, d) an inhibitorof osteoclast proton ATPase, e) a parathyroid hormone analog, and f) acathepsin K inhibitor; and mixtures thereof.
 8. The composition of claim7 wherein said organic bisphosphonate or pharmaceutically acceptablesalt or ester thereof is alendronate monosodium trihydrate.
 9. A methodof inhibiting a condition selected from the group consisting of boneresorption, restenosis, angiogenesis, diabetic retinopathy, maculardegeneration, inflammatory arthritis, and tumor growth comprisingadministering to a mammal in need thereof a therapeutically effectiveamount of a compound according to claim
 1. 10. The method of claim 9wherein the condition to be inhibited is bone resorption.
 11. A methodof inhibiting bone resorption in a mammal in need thereof, comprisingadministering to the mammal a therapeutically effective amount of thecomposition of claim
 5. 12. A method of inhibiting bone resorption in amammal in need thereof, comprising administering to the mammal atherapeutically effective amount of the composition of claim
 7. 13. Amethod of treating or preventing osteoporosis which comprisesadministering to a mammal in need of such treatment or prevention atherapeutically or prophylactically effective amount of a compound ofclaim
 1. 14. A method of treating or preventing osteoporosis whichcomprises administering to a mammal in need of such treatment orprevention a therapeutically or prophylactically effective amount of acompound of claim 1 in combination with an effective amount of anorganic bisphosphonate or a pharmaceutically acceptable salt or esterthereof.
 15. The method of claim 14 wherein said organic bisphosphonateor pharmaceutically acceptable salt or ester thereof is alendronatemonosodium trihydrate.